1. Field of the Invention
The present invention relates to a gene-modified T-cell for anticancer T-cell therapy and a composition for anticancer therapy comprising the same.
2. Description of the Related Art
The most fundamental and effective method for the treatment of cancer is surgical excision. However, it is not easy to remove residual tumor or metastatic foci by surgical excision alone, and thus various treatment methods such as chemotherapy, radiation therapy and the like have been used in combination with surgical excision. Despite the development of such treatment methods, the effective treatment of multiple metastases or other biochemical recurrence appearing after surgical excision is still difficult and remains as an important problem to be solved by medical science.
As a good method for the treatment of tumor foci or invisible micro-foci in various organs, immunotherapy based on the in vivo immune system has recently received attention. Robert Schreiber et al. reported a significant increase in the incidence of cancer in lymphocyte deficient mice or IFN-γ (effector cytokine)-knockout mice, suggesting that the development of cancer is inhibited by the immune system, particularly lymphocytes (Nature, 2001, vol. 410; 1107-1111). Particularly, there were many reports that specific antibodies or T-cells capable of recognizing tumor-associated antigens are present in vivo, suggesting that the applicability of anticancer immunotherapies is very high.
Therapies that have recently received the most attention among immunotherapies include cell therapies in which immune cells such as dendritic cells, natural killer cells (NK cells), or T-cells are injected directly into patients. Among them, T-cell therapy produces visible outcomes. The fundamental concept of T-cell therapy is that cancer antigen-specific T-cells are isolated from a patient, then cultured in vitro in large amounts, and are returned to the blood of the patient to attack cancer cells (Nat. Rev. Immunol. Vol. 6, 383). In other words, a small number of cancer-specific T-cells are cultured in vitro to increase the numbers thereof and are then used for cancer treatment.
In particular, the antigen specificity and tissue penetration ability of T-lymphocytes are expected to make it possible to effectively remove tumor foci present in various places. T-cells can penetrate tissue via extravasation to specifically kill antigen-expressing cells, and thus can penetrate various cancer tissues to remove cancer cells. Thus, in recent years, cancer therapies based on these anticancer T-cells have been actively developed.
Cell therapy based on T-cells has been attempted for the past 20-30 years by Dr. Steve Rosenberg et al. (NUH), named LAK (lymphokine-activated killer) or TIL (tumor-infiltrating lymphocyte) cell therapy, but the effectiveness thereof has been demonstrated in a limited number of cases. However, in recent years, it was reported that an attempt to deplete a patient's lymphocytes before injection of T-cells showed a response rate of 50%, including complete remission in metastatic melanoma patients, and thus this attempt was evaluated to make an important breakthrough for adoptive T cell transfer therapy (Science 2002, 298(5594):850-4; J. Clin. Oncol. 2005, 23(10):2346-57). It is presumed that lymphocyte depletion before injection of T-cells makes space for the proliferation of T-cells to be injected and has the effect of removing regulatory T-cells (i.e., an inhibitor of T-cell activator). This response rate is the highest among those in immunotherapies attempted to date and indicates the prospect of further development of this therapy.
By virtue of the success of cancer antigen-specific T-cell therapy, in recent years, there have been active studies on genetically engineered T-cell therapy in which isolated T-cells are genetically engineered to enhance the nature and efficacy thereof, and then injected into the patient. The concept of genetically engineered T-cell therapy is that cancer antigen-specific T-cells isolated from a patient are proliferated and then injected into the patient using a gene expression retrovirus after transduction. This concept is beyond the laboratory stage and is now in clinical trials in many cases.
However, in order to effectively remove cancer cells by cancer therapy using such cancer-specific T-cells, obstacles including the immune tolerance or suppression ability of cancer cells should be overcome. Development of cancer in normal mice or persons who have immunity, despite the tumor suppression ability of the immune system, means that cancer cells have resistance to the immune system, or immune evasion.
The reason why the immune tolerance of cancer cells is created has not yet been clearly established, and various hypotheses for the reason(s) have been presented, but the hypothesis that cancer cells induce tolerance to anticancer lymphocytes is valid. In other words, this hypothesis is that T-lymphocytes that can recognize and disrupt cancer cells are indeed present in the human body, but cancer cells or microenvironments surrounding cancer cells inactivate these anticancer T-cells.
Indeed, it was reported that cancer antigen-specific T-cells collected from melanoma patients secrete IFN-γ when they are stimulated with the cancer antigen Melan-A peptide, whereas cancer antigen-specific T-cells collected from cancer tissue or cancer tissue lymph nodes are in an inactivated state in which they cannot secrete IFN-γ even when they are stimulated with the antigen. This suggests that the peripheral blood of cancer cells has T-cells capable of recognizing and responding to cancer cells, but these T-cells are locally inactivated (e.g., tolerant) when they enter cancer tissue.
In other words, when T-cell tolerance to cancer cells is removed, the cancer cells can be effectively removed. Thus, it is an important prerequisite for anticancer immunotherapy to break immunological tolerance to cancer cells to activate anticancer lymphocytes. Various studies focused on the removal of T-cell tolerance to cancer cells have been conducted. Particularly, studies have been actively conducted to identify a receptor or protein involved in T-cell tolerance to cancer cells, and to remove or inhibit the function thereof or increase treatment effects using an antagonist or antibody against the receptor.
A receptor that is typically known to be involved in T-cell tolerance is Cytotoxic T-Lymphocyte-Associated Protein 4, or T-Lymphocyte Antigen 4 (CTLA4), also called CD152. CTLA4, a member of the superfamily of immunoglobulin, is expressed on the surface of T-cells and transduces an inhibitory signal to T-cells. The induction of tolerance by T-cell inactivation by the CTLA4 protein was confirmed by observation of severe lymphoproliferative disease and autoimmune disease in CTLA4-knockout mice.
CTLA4 has a sequence similar to that of the T-cell costimulatory protein CD28 and binds to CD80 and CD86, also called B7 of antigen-presenting cells, competitively with CD28. When it binds to B7, CTLA4 transduces an inhibitory signal, and CD28 transduces a stimulatory signal. In other words, when B7 binds to CTLA4, the activation of T-cells is inhibited, and when B7 binds to CD28, the activation of T-cells is induced.
Another protein that is involved in T-cell tolerance is PD1. It is known that PD1 is expressed on the surface of T-cells and binds to PD-L1 to inhibit the activation of T-cells. It is known that PD-L1, a family member having a structure similar to that of CD28, is expressed mainly on the surface of immune cells, including T-cells, B-cells, macrophages and dendritic cells, and is also expressed in some non-lymphoid cells such as cardiac vascular endothelial cells.
It is known that autoimmune disease naturally occurs in PD1-knockout mice and a native signal is transduced into T-cells by PD1 stimulation, suggesting that the inhibition of T-cell activity by the interaction between PD-L1 and PD1 is very important in immune tolerance.
However, in recent years, an increase in the expression in PD-L1 in many kinds of cancer tissues was observed (Nat. Med. 2002 August; 8(8):793-800), and it was reported that treatment with a blocking antibody against PD-L1 results in an increase in anticancer immunity (Proc. Natl. Acad. Sci. 17; 99(19):12293-7). This demonstrates that PD-L1 acts as an immune inhibitor on the surface of cancer cells.
Thus, it is expected that inhibition of receptors or proteins, such as CTLA4 or PD1, which are involved in T-cell immune tolerance can provide anticancer effects. Accordingly, studies on T-cell immunotherapy as a strategy for inhibiting the activity CTLA4 or PD1 using its antibody have been actively pursued.
In particular, it was clinically demonstrated that the anti-CTLA4 antibody Ipilimumab developed by Bristol-Myers Squib (BMS), or other similar companies, shows an anticancer effect against metastatic melanoma by inhibiting immune tolerance. Thus, this antibody was approved by the FDA in 2011 and is now marketed. Also, it is known that BMS and other companies are performing clinical trials for a complete humanized anti-PD1 antibody.
However, it is known that the use of anti-CTLA4 antibody or anti-PD1 antibody disrupts not only anticancer T-cells, but also T-cell tolerance to self-antigen, due to the systemic inhibition of CTLA4 or PD1, resulting in fatal side effects.
Thus, for actual clinical application of cancer antigen-specific T-cell therapy, there is an urgent need for the development of technology capable of inhibiting a T-cell tolerance signaling system for CTLA4 or PD1 only in anticancer T-cells.
Accordingly, the present inventors attempted to increase the activity of anticancer T-cells by competitively inhibiting the function of T-cell endogenous CTLA4 as a result of genetically engineering anti-cancer T-cells so as to express a CTLA decoy receptor that is a mutant CTLA lacking the intracellular inhibitory signaling domain of CTLA4. However, in the case of such genetically engineered T-cells, there was a problem in that the CTLA decoy receptor competitively inhibits the binding between CD28, that induces T-cell activation, and the ligand B7, ultimately resulting in the inhibition of T-cell activation, even though the CTLA decoy receptor may somewhat solve the T-cell tolerance problem, because an inhibitory signal is not transduced into cells even when the CTLA decoy receptor binds to the ligand B7.
Accordingly, the present inventors have designed an anticancer T-cell genetically engineered so as to express a CTLA4-CD28 chimera protein, which includes CTLA4 lacking its intracellular inhibitory signaling domain and the intracellular stimulatory signaling domain of CD28 protein, fused thereto (see FIG. 1). Using this designed anticancer T-cell, the present inventors have found that when a ligand binds to CTLA4, a T-cell inhibitory signal caused by the binding between CTLA4 and the ligand is converted to a stimulatory signal by the action of the intracellular stimulatory signaling domain of CD28 in the CTLA4-CD28 chimera protein, T-cell tolerance to cancer cells can be overcome, anticancer effects of the T-cell can be greatly enhanced by the activation thereof, and a side effect such as the development of autoimmune disease caused by the systemic inhibition of CTLA4 activity can be avoided, suggesting that the T-cell can be used for ideal T-cell immunotherapy. In addition, the present inventors have found that a T-cell genetically engineered so as to express a PD1-CD28 chimera protein that includes PD1 lacking its intracellular domain known to be involved in T-cell tolerance, similar to CTLA4 in the CTLA4-CD28 chimera protein, also increases T-cell activation, suggesting that the T-cell can be used for ideal T-cell immunotherapy.